The objective of this research proposal is to purify erythropoietin from crude urine preparations in quantities sufficient for biochemical studies and for clinical therapeutic trials. To this end we plan to exploit differences in the carbohydrate moieties of urine proteins to isolate erythropoietin by utilizing affinity chromatography with agarosebound plant and invertebrate lectins. Two lectins, wheat germ agglutinin and phytohemagglutinin (PHA, P form) have been identified which specifically bind erythropoietin. Four other lectins, concanavalin-A, ricinus communis-120, soybean agglutinin, and limulin, which do not bind erythropoietin, bind substantial quantities of other urine proteins. Sequential chromatography using these lectin-agarose derivatives is capable of separating erythropoietin from over 90% of the contaminating proteins in crude preparations of the hormone. In order to improve the efficiency of this technique, we plan to investigate the use of purified PHA and four other additional lectins, peanut agglutinin, lens culinaris A and B, and fucose binding lectin as affinity adsorbents. The use of other adsorbents such as th agarose derivative of alkyl succinic anhydride which specifically binds albumin, a major contaminant of crude erythropoietin preparations, will also be evaluated. Affinity chromatography in conjunction with conventional separation techniques should permit the production of a highly purified hormone preparation in high yield. In order to make the hormone suitable for human use, we plan to remove endotoxin from our preparations by adsorption with the amebocyte lysate of the horseshoe crab. The homogeneity of purified erythropoietin will be assessed by gel electrophoresis, electrofocusing, and amino-terminal end group analysis. Purified erythropoietin will be used to develop an immunoassay for the hormone.